Placenta 31 (2010) 106–112
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Feto-maternal interactions in pregnancies: Placental microparticles activate peripheral blood monocytes M. Messerli a, K. May b, c, S.R. Hansson b, H. Schneider d, W. Holzgreve a, S. Hahn a, C. Rusterholz a, * a
Laboratory for Prenatal Medicine, Department of Biomedicine/University Hospital Basel, Spitalstrasse 21, CH-4031 Basel, Switzerland Department of Obstetrics and Gynecology, Lund University, University Hospital Lund, Lund, Sweden c Department of Clinical Pharmacology, Ernst Moritz Arndt University of Greifswald, Germany d Department of Obstetrics and Gynecology, University of Bern, Bern, Switzerland b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 23 November 2009
Normal pregnancy is associated with a systemic maternal inflammatory reaction, including the activation of peripheral blood monocytes. This reaction is exaggerated in pre-eclampsia, a severe placentadependent disorder of pregnancy specific to humans. It has been suggested that placental syncytiotrophoblast membrane microparticles (STBM), which are released into the peripheral blood, may contribute to the maternal response. The aim of this study was to investigate the inflammatory properties of STBM generated by four different approaches on primary human monocytes in vitro. Cellular viability, phenotype and functional response were analysed. STBM isolated by mechanical dissection and STBM generated from villous explant cultures incubated in hypoxic conditions had only minor influences on the monocytic phenotype and failed to induce a proinflammatory response. By contrast, STBM washed from the maternal side of a placental cotyledon and STBM shed by explants cultured in air upregulated cell surface expression of the adhesion molecule CD54 and induced the production of interleukin (IL)-8, IL-6 and IL-1b. Cytokine production was time- and dose-dependent. Our study, therefore, suggests that monocyte activation in normal pregnancy and pre-eclampsia may be induced by STBM released by the placenta. The higher amounts of STBM circulating in maternal blood in pre-eclampsia might lead to the excessive maternal inflammatory reaction. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Pregnancy Placental microparticles Monocytes Feto-maternal interactions
1. Introduction Pre-eclampsia is a placenta-dependent disorder specific to human pregnancies which represents a major cause of maternal and fetal morbidity and mortality worldwide [1]. The maternal symptoms, which usually arise in the second half of pregnancy, are hallmarked by hypertension, proteinuria and oedema. An excessive systemic maternal inflammatory response, including a dysfunctional endothelium and the activation of the innate immune system, is thought to be at the basis of the clinical manifestations of the disease [2]. Pregnancy itself has been described as a state of controlled mild inflammation [3]. Although studies have given controversial results about the proportion of peripheral blood monocytes in pregnant compared to non-pregnant women [4,5], they showed a progressive monocytic activation in the circulation of pregnant women [6–8]. Monocytes displayed an enhanced phagocytic rate with peak levels
* Corresponding author. Tel.: þ41 61 265 7088; fax: þ41 61 265 9399. E-mail address:
[email protected] (C. Rusterholz). 0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2009.11.011
in the third trimester, a significantly higher baseline of reactive oxygen species, changes in the expression of the cell surface adhesion molecules CD54 and CD11c, and an increased intracellular production of proinflammatory cytokines [6–11]. Monocytes from pre-eclamptic patients demonstrated a further increase in basal intracellular reactive oxygen species, a higher synthesis of interleukin (IL)-1b, IL-6 and IL-8 and more pronounced changes in the expression profile of cell surface markers, such as CD11b and CD14, compared to monocytes from healthy pregnant women [9,11–13]. Monocyte-derived microparticles were also more elevated in preeclamptic patients compared with pregnant controls, reflecting the activation of their parental cells in pre-eclampsia [14]. Until now, the cause of the inflammatory response in both normal and pre-eclamptic pregnancy is unknown. Beside placental cytokines and anti-angiogenic factors, a candidate trigger of the systemic inflammatory response in pregnancy is apoptotic, apo-necrotic or necrotic debris shed from the syncytiotrophoblast into the maternal blood [15–17]. This debris consists of trophoblast cells and large cellular fragments, such as multinucleated syncytial knots, which are trapped and degraded by alveolar macrophages in the maternal lungs, and syncytiotrophoblast
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membrane microparticles (STBM) [18–21]. It has been suggested that STBM are constantly shed from the placental syncytium into the intervillous space, from which they circulate into the maternal peripheral blood, where they will come in direct contact with the maternal endothelium and leukocytes. Significantly increased amounts of STBM have been found in the circulation of women with pre-eclampsia [21,22], consistent with the increased placental damage and dysfunction observed in this pathological condition. As placental derived microparticles only account for less than 6% of the total number of microparticles in the blood of normotensive pregnant women, it is very difficult to isolate pure and adequate amounts of placental microparticles from maternal blood [23]. Several in vitro and ex vivo approaches for STBM generation have been described [24]. We and others provided evidence that artificially prepared STBM affect the viability and/or function of endothelial cells and neutrophils in vitro [24–28]. These studies also revealed that the mode of preparation of the STBM differentially affected the response of the cellular target. Less is known about the possible effects of STBM on monocytes. One recent study showed that STBM isolated ex vivo from the maternal perfusion of a placental cotyledon induces the intra-monocytic production of the TH1 cytokines TNF-a and IL-12p70, thereby strongly suggesting that placental microparticles may play a role in the development of the inflammatory state in pregnancy [29]. In this study, we generated STBM by four different methods and analysed their effect on changes in monocytic cell surface expression and cytokine secretion.
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was measured with the advanced protein assay reagent (Cytoskeleton Inc., Denver, CO) according to the manufacturer’s recommendations. The optical density (OD) was read at 595 nm in a Spectramax 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). In all preparations placental alkaline phosphatase (PLAP) could be detected, confirming the presence of microparticles from the syncytiotrophoblast. Moreover, aliquots of supernatants from explant cultures and postperfusion media were incubated at 37 C for 48 h to confirm the absence of bacterial growth and, thus, contamination of these media with endotoxin. 2.2. Isolation of human monocytes Forty millilitres of venous blood from healthy male donors, collected in EDTAcontaining tubes, were diluted in PBS supplemented with 2 mM EDTA and the mononuclear cells (PBMCs) were separated by density gradient centrifugation on Histopaque (Sigma, Saint Louis, MO). PBMCs were washed twice with PBS/2 mM EDTA. The remaining erythrocytes were lysed with red blood cell (RBC) lysis solution (Qiagen, Valencia, CA). Monocytes were isolated by negative selection using the human Monocyte Isolation Kit II and magnetic cell separation (Miltenyi Biotec Inc., Auburn, CA), according to the manufacturer’s protocol. 2.3. Co-cultures of monocytes and STBM
2. Materials and methods
Monocytes were cultured at 5 105 cells/ml in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 10% FCS (Amimed, Allschwil, Switzerland), 4 mM glutamine (Gibco, Grand Island, NY), 100 U/ml penicillin/streptomycin (Gibco, Grand Island, NY). STBM were added in different concentrations, as indicated in the figure legends. STBM preparations were tested individually on at least 2 different monocyte populations to exclude a donor-specific response. As a positive control of the monocyte response, cells were treated with lipopolysaccharide (LPS) (Sigma, Saint Louis, MO) from gram-negative bacteria. In some experiments, STBM were pre-treated with mouse anti-human IL-8 antibody (R&D Systems Inc., Minneapolis, MN), as illustrated in Fig. 4, for 15 min before co-culture with monocytes. Alternatively, the cells were cultured with 1 ng/ml, 10 ng/ml and 100 ng/ml of recombinant human IL-8 (Sigma, Saint Louis, MO). Monocytes and culture supernatants were separately harvested after 4 h or 16 h incubation at 37 C in air/5% CO2.
2.1. Preparations of STBM
2.4. Cell viability
This study was approved by the Cantonal Institutional Review Board of Basel, Switzerland, and the Ethical Committee Review Board for studies in human subjects of Lund, Sweden, and written informed consent was received in all cases. Human term placentas from uncomplicated pregnancies were obtained from the University Women’s Hospital Basel, or the Department of Obstetrics and Gynecology, Lund University Hospital, immediately after elective caesarean section or vaginal delivery. STBM were generated by four different approaches, indifferently of the mode of delivery.
Following co-culture, monocytes were harvested, washed, resuspended in 100 ml complete RPMI-1640 (Gibco, Grand Island, NY) culture medium and transferred into 96-well plates. Ten microlitres of WST-1 reagent (Roche Diagnostics GmbH; Mannheim, Germany) were added to each well. As a negative control, the same volume of fresh culture medium and WST-1 reagent were used. The plate was incubated for 2 h at 37 C in air/5% CO2 and analysed on the Spectramax 250 microplate spectrophotometer at 450 nm, corrected by the reference wavelength of 600 nm.
2.1.1. In vitro explant cultures (eS20 and eS3) The placental villous tissue was cut into small pieces (2–4 mm3) free of any visible vessels and washed three times in phosphate buffered saline (PBS) to remove blood cells. Explants were cultured in 10 cm-diameter dishes (Corning, NY) in Dulbecco Modified Eagle’s Medium (DMEM): F12 Nutrient Mixture (1:1) (Gibco, Grand Island, NY) supplemented with 10% FCS (Amimed, Allschwil, Switzerland), 1 antibiotic/antimycotic (Gibco, Grand Island, NY) 25 IU/ml heparin (B. Braun Medical AG, Sempach, Switzerland) and 50 U/ml aprotinin (Fluka, Buchs, Switzerland) for 72 h at 37 C. Incubation was performed in 20% O2/5% CO2 for 5 placentas (eS20), or in 3% O2/5% CO2 (hypoxia) for 3 placentas (eS3). 25 mg/ml of vitamin C (Sigma, Saint Louis, MO) was added to the cultures incubated in 20% O2/5% CO2 to preserve villous tissue from elevated programmed cell death or necrosis [30]. The same placenta was used for cultures under both oxygen conditions.
2.5. Flow cytometry To block unspecific binding through Fc receptors (FcRs), monocytes were preincubated with purified human IgG (Sigma, Saint Louis, MO), diluted to a working concentration of 200 mg/ml in PBS/2 mM EDTA/0.5% BSA for 5 min at 4 C. Specific antibody stainings were performed for 15 min at 4 C with ready to use concentrations of FITC-conjugated anti-CD14, PE-conjugated anti-CD54 and APC-conjugated anti-CD11a (BD Pharmingen, San Jose, CA) antibodies. After washing, the stained cells were resuspended in PBS/2 mM EDTA/0.5% BSA and 10,000 events were acquired on a Dako Cyan flow cytometer (Beckman Coulter, Fullerton, CA) and analysed with the Summit software.
2.1.2. Mechanical dissection (mS) The villous tissue from 4 placentas was separately dissected using the protocol described in [24]. In brief, small fragments of villous tissue were extensively washed in PBS and stirred in 0.15 M NaCl supplemented with 400 U/ml penicillin/streptomycin (Gibco, Grand Island, NY) overnight at 4 C.
IL-8, IL-6 and IL-1b were quantified with commercial DuoSetÒ ELISA Development Kits (R&D Systems Inc., Minneapolis, MN) following the manufacturer’s instructions. OD was read on the Spectramax 250 microplate spectrophotometer at 450 nm with the wavelength correction of 562 nm. Samples and standard were measured in duplicates and mean values were calculated.
2.1.3. Placental dual perfusion (pS) A suitable cotyledon was set up for separate dual perfusion of the maternal (intervillous space) and the fetal compartment (villous vasculature), without recirculation at flow rates of approximately 12 and 6 ml/min, respectively, using a welldescribed perfusion system [31]. Particulate matter was collected with a 30 min wash of the maternal side of the dually perfused, placental cotyledon from three placentas. In all instances, STBM were purified by a three-step centrifugation at 4 C: 1000g for 10 min, 10,000g for 10 min, and 60,000g for 90 min. The final pellets were washed with PBS, resuspended in PBS containing 5% sucrose and stored in aliquots at 20 C until use. The protein content of the different STBM preparations
2.7. Caspases activity assay
2.6. Enzyme-linked immunosorbent assay (ELISA)
Whole protein extracts were obtained by homogenizing the explants in 50 mM Tris pH 7.6, 150 mM NaCl, 1 protease inhibitor cocktail, 1% Triton X-100 and 0.5 mM PMSF, with a Polytron PT 1200 E tissue homogenizer (Kinematica AG, LittauLucerne, Switzerland). An adaptation of the fluorimetric homogeneous caspases assay (Roche Diagnostics GmbH; Mannheim, Germany) was used to assess activated caspases 2, 3, 6, 7, 8, 9 and 10. In brief, protein extracts were diluted 1/10 in the incubation buffer, 1 volume of the substrate working solution was added and the reaction was incubated at 37 C for 24 h. The cleavage of the substrate by active caspases was measured at Ex 485 nm/Em 538 nm. The cut off value was set at
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530 nm. Relative fluorescence units (RFUs) were measured on a Spectramax Gemini spectrofluorometer (Molecular Devices, Sunnyvale, CA), using the Softmax Pro software (Molecular Devices, Sunnyvale, CA). The results are presented as RFU/mg of proteins. 2.8. Lactate dehydrogenase (LDH) cytotoxicity assay The release of LDH into the culture medium was assessed relative to a positive control, made from a villous explant lysed in 1% Triton X-100, using an adaptation of the colorimetric lactate dehydrogenase based toxicity assay (Sigma, Saint Louis, MO). In brief, culture supernatants were diluted 1/3 in the assay mixture consisting of equal amounts of substrate, enzyme and dye solutions. The reaction was stopped with 1 N HCl and the OD was measured at 490 nm in a Spectramax 250 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Percent necrotic cell death was calculated as (OD of sample/OD of Triton X-100-lysed explant) 100%. 2.9. Statistical analysis Data were compared with the Mann–Whitney U test using the statistical analysis software SPSS (Statistical Package for the Social Sciences; Chicago, IL, USA). The p < 0.05 was considered statistically significant.
3. Results 3.1. STBM do not affect the viability of human monocytes Purified populations of monocytes generally contained >94% CD14þ cells, as confirmed by flow cytometry (data not shown). These cells were incubated with STBM prepared by four different approaches, namely from in vitro cultures of explants from villous tissue incubated at 20% O2 (eS20) or 3% O2 (eS3), or from washes of the maternal side of ex vivo dually perfused placental
lobe (pS), or by mechanical dissection of villous tissue (mS). None of the STBM preparations substantially altered monocytic viability as observed in the flow cytometer analysis (Fig. 1a). Furthermore, cell viability was also routinely quantified by a colorimetric assay, which measures mitochondrial dehydrogenase activity, and was always >80% relative to the untreated monocytes that were cultured in parallel to the STBM-treated cells (data not shown). 3.2. STBM differently alter the expression profile of cell surface markers on human primary monocytes In order to investigate the effect of STBM on the phenotypic activation status of monocytes, we measured the cell surface expression of CD14, CD54 (intercellular adhesion molecule 1/ ICAM-1) and CD11a (alpha L integrin), which have been previously shown to be differently expressed on monocytes of pregnant and pre-eclamptic women compared to non-pregnant controls [8,9]. Neither the well known monocyte activator LPS, nor the four different STBM populations altered CD14 expression relative to untreated cells following a 16-h co-incubation (Fig. 1b and c). In contrast, stimulation of monocytes with LPS led to a significant increase in the median fluorescent intensity (MFI) of CD54 and a significant decrease in the expression of CD11a. Incubation with eS20 and pS also significantly enhanced monocytic expression of CD54, whereas eS3 and mS did not alter CD54 MFI. Cell surface expression of CD11a was significantly reduced by pS and mS.
Fig. 1. Expression of CD14, CD54 and CD11a on monocytes co-incubated with different STBM populations. (A) Representative forward scatter/side scatter (FSC/SSC) dot plots of monocytes, left untreated or incubated with 300 mg/ml STBM or 1 mg/ml LPS for 16 h. (B) Representative histograms of CD14, CD54 and CD11a expression on R1 gated cells cocultured with the indicated STBM preparations. (C) Differences in median fluorescent intensity (MFI) of CD14, CD54 and CD11a stimulated by LPS or the different STBM populations. Bars illustrate mean SEM of at least 2 experiments with STBM generated from 3 to 5 different placentas for each condition (see Section 2). Data were analysed using the Mann– Whitney U test. *p value < 0.05, **p value < 0.01.
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3.3. eS20 and pS induce the secretion of proinflammatory cytokines by human monocytes Since the changes in cell surface expression of adhesion molecules upon incubation with STBM may indicate monocytic activation, we next investigated whether STBM could stimulate the secretion of the chemokine IL-8 and the proinflammatory cytokines IL-1b and IL-6. Monocytes cultured for 16 h in the absence of treatment produced basal levels of IL-8, but did not secrete detectable levels of proinflammatory cytokines (Fig. 2a). As expected, stimulation with LPS enhanced IL-8 secretion and induced the production of IL-1b and IL-6 compared to the untreated cells. Incubation with eS20 led to a comparable statistically significant increase in IL-8 release, while at the same dose of STBM protein, pS triggered a threefold higher secretion of IL-8 than eS20. Both eS20 and pS also stimulated IL-6 and IL-1b production, although to varying degrees. Whereas the cellular response to eS20 remained modest and was always lower than that induced by LPS, the stimulatory effect of pS on cytokine expression was extreme. IL-6 and IL-1b secretions were, respectively, 3.5-fold and 2.6-fold higher upon incubation with pS than following stimulated with LPS (Fig. 2a, medium and lower panels). In contrast, eS3 and mS did not induce monocytic release of IL-8, IL-6 or IL-1b. The presence of endotoxin, which could significantly influence the results, in microparticle preparations of eS20 and pS is very unlikely as there was no evidence of bacterial contamination in the
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culture supernatants and post-perfusion media (data not shown). Furthermore, soluble endotoxins are expected to be eliminated upon isolation of the STBM through centrifugation. 3.4. STBM-induced cytokine secretion is dose- and time-dependent The secretion of proinflammatory factors stimulated by eS20 and pS was dependent on the concentration of microparticles, which was evaluated by their protein content. Increasing doses of eS20 and pS mediated a steady rise in IL-8 production (Fig. 2b, upper panel). IL-6 secretion in response to eS20 was also dose-dependent (Fig. 2b, medium panel). In contrast, the levels of IL-6 already reached a plateau at the lowest dose of pS and did not increase further. Moreover, pS induced a higher cytokine response than eS20 at all doses of STBM tested. pS also stimulated IL-1b secretion in a dosedependent manner (Fig. 2b, lower panel). Cytokine production in response to STBM was also timedependent. While IL-6 secretion increased steadily from 4 h to 16 h following stimulation, IL-1b release reached maximum levels already at 4 h and was sustained with longer incubation (Fig. 2c). 3.5. eS20 preparations contain IL-8 The placental syncytiotrophoblast produces many proinflammatory cytokines, which might be associated with the microvesicles when the latter shed off from the syncytiotrophoblast membrane. In particular, we previously showed that STBM
Fig. 2. STBM-mediated secretion of cytokines by monocytes. (A) Quantification of IL-8, IL-6 and IL-1b secreted by monocytes in response to the different STBM populations. Cells were left untreated or co-incubated for 16 h with 300 mg/ml STBM or 100 pg/ml LPS. (B) Dose-dependent production of IL-8, IL-6 and IL-1b induced by pS and eS20. (C) Timedependent secretion of IL-6 and IL-1b upon treatment with eS20. Data are presented as mean SEM of 2 or more experiments. eS20-associated IL-8 was deduced from total levels of IL-8 measured in the co-cultures (in order to evaluate the monocytic contribution). *p value < 0.05, **p value < 0.01.
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prepared from villous explant culture incubated in air, contained detectable amounts of IL-8 [28]. As cytokines influence many aspects of monocyte activation, we analysed the different STBM preparations for the presence of proinflammatory factors. None of the STBM preparations contained detectable levels of IL1b or TNF-a (data not shown). IL-6 was either undetectable or present at very low concentrations (Table 1). IL-8 was similarly absent or low in pS, eS3 and mS. However, high levels of IL-8 were reproducibly associated with preparations of eS20 (Table 1). 3.6. IL-8 is not responsible for eS20-mediated cytokine secretion Although IL-8 has never been described as a stimulus for monocytes, we confirmed that it was not responsible for the eS20-mediated activation of the cells. First, monocytes, which were stimulated with three different concentrations of human recombinant IL-8 for 16 h did not produce IL-6 (data not shown). Furthermore, IL-6 secretion in response to eS20 was not significantly affected by the presence of an anti-human IL-8 blocking antibody (Fig. 3). 3.7. The disparate activities of eS20 and eS3 cannot be explained by variation in their apo-necrotic properties In order to determine if the distinct responses of the monocytes to eS20 and eS3 were associated with a more apoptotic or necrotic way of release, we assessed caspases’ activity and the release of LDH in placental explant cultures incubated in 20% or 3% oxygen. Villous tissue explants cultured at 3% O2 showed lower caspases activity but higher LDH release relative to the explants incubated at 20% O2 (Fig. 4a and b). However, these changes failed to reach statistical significance. 4. Discussion This study examined the effects of in vitro prepared STBM on human peripheral blood monocytes through an investigation of cellular viability, phenotype and function. Our data show that different STBM populations trigger various responses in these cells without affecting cell viability. While mS and eS3 had no or only minor influences on the monocytic phenotype and function, pS and eS20 induced a proinflammatory response. That the monocytic response varied according to the mode of preparation of the STBM is not surprising in the light of previous findings with other cell types. For instance, it was shown that mS triggered endothelial cell detachment from the collagen matrix and apoptosis, whereas eS20 and pS induced a partial inhibition of endothelial cell proliferation, but no apoptosis [24,25]. Furthermore, eS20 and mS significantly inhibited, whereas pS enhanced, the proliferation of T lymphocytes induced with phorbol ester and Ca2þ ionophore [32]. It was suggested that this may reflect the more apoptotic or necrotic nature of the differentially generated STBM, providing an in vitro model to study the role of placental debris in normal pregnancy and preeclampsia. However, the functional differences between eS20 and eS3 do not lie on a fundamentally different apo-necrotic shedding, as our analysis shows similar tissue apoptosis and necrosis in
Table 1 The presence of STBM-associated cytokines was measured by ELISA. All results are shown as mean SEM.
eS20 pS eS3 mS
IL-8 (ng/mg STBM)
IL-6 (ng/mg STBM)
44.0 3.2 0.0 0.7 0.3 0.07 0.03
0.13 0.08 0.0 0.07 0.06 0.0
Fig. 3. STBM-induced IL-6 secretion in the presence of IL-8 blocking antibody. Three hundred mg/ml of eS20 were pre-treated with the indicated concentrations of mouse anti-human IL-8 antibody, before co-culture with monocytes for 16 h. All data are presented as mean SEM of 2 experiments.
explants cultured at 20% or 3% O2. Further characterization of these microparticles will be required. Here, we show that monocytes treated with pS and eS20 acquired an activation-like phenotype and secreted proinflammatory immune mediators. Up-regulation of the adhesion molecule CD54 is of interest in regard of a previous report documenting a significant increase in CD54 expression on peripheral blood monocytes from third trimester pregnancies [8]. Additionally, plasma from normal pregnant and pre-eclamptic women induced the expression of CD54 on a monocytic cell line in vitro [33]. Our results suggest that placental microparticles could be the mediators of these observations. pS and eS20 also stimulated the production of the proinflammatory molecules IL-8, IL-6 and IL-1b. We chose to analyse these factors because their intracellular expression is enhanced in peripheral blood monocytes of pre-eclamptic women [13]. Furthermore, they are also present in elevated concentrations in the serum of pre-eclamptic patients [34–36]. Our results are also in line with a recent report, which showed that STBM prepared by dual perfusion of a placental cotyledon, but not STBM generated by mechanical dissection of villous tissue, induced a functional response in primary monocytes in vitro, as measured by the increased intracellular production of TNF-a and IL-12p70 [29]. However, in this study, STBM were incubated with the whole peripheral blood mononuclear cell (PBMC) fraction, leaving open the possibility that the up-regulation of cytokine expression in monocytes could be indirect, for instance by cell-cell contact between monocytes and STBM-activated third-party cells, or alternatively, through stimulation by a soluble mediator produced by the latter. Our findings allow us to conclude that STBM are capable of directly inducing monocytic cytokine production. The monocytic response was time-dependent. As expected, IL-6 secretion increased steadily with incubation time, whereas the secretion of IL-1b was rapid and sustained. This observation is in agreement with the knowledge that IL-1b is a mediator of acute inflammation, whereas IL-6 is a secondary pleiotropic regulator of inflammatory responses. IL-1b is stored as an inactive precursor molecule in the cytoplasm, which is cleaved and released as a proactive form in the extra-cellular compartment immediately after stimulation [37]. Our results also show that monocyte activation in response to STBM was dose-dependent. Moreover, at identical protein concentration, the response stimulated by pS was much higher than that induced by eS20. This could indicate that these two preparations of microparticles are functionally different. On the other hand, this might also reflect a technical limitation of our work. To conform to previous studies, we used the STBM-associated protein concentration to quantify the STBM populations [24,29].
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Fig. 4. Expression of activated caspases and release of LDH by villous tissue explants. Villous explants were cultured for 72 h at 37 C in either 20% O2 or 3% O2. The same placentas were used for cultures under both oxygen conditions. (A) The levels of active caspases in protein extracts of villous explants are expressed as relative fluorescence units (RFU)/mg of tissue protein. (B) The percentage of LDH released in the culture medium is indicated relative to the LDH activity from an explant that was lysed in detergent. Bars are presented as mean SEM of 3 explant cultures.
However, this is a surrogate measurement for microparticle numbers as the protein content of differentially prepared STBM may vary. Another aspect that needs to be considered when interpreting the current results is that, although the presence, in our STBM preparations, of syncytial-membrane microparticles could be confirmed by the detection of syncytiotrophoblast-specific PLAP (data not shown), we cannot exclude the possible occurrence of microparticles derived from non-trophoblastic elements of the villous tissue or from residual maternal blood cell debris. Notwithstanding these limitations, we would like to suggest that STBM are potent activators of monocytes. It is likely that one of the molecules through which the activation signal triggered by STBM is conveyed, is the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). NF-kB is a master transcription factor involved in inflammatory pathways which regulates the expression of several genes, including CD54, IL-8, IL-6 and IL-1b [38]. NF-kB also acts as a cell survival factor by inducing the transcription of anti-apoptotic regulators, such as GADD45b (Growth arrest and DNA-damage-inducible 45b) or BclxL (Basal cell lymphoma-extra large). Further investigations will be required to confirm a role for this factor in the STBM-mediated activation of monocytes. Another open question that needs to be addressed in future research pertains to identifying which cell surface molecule(s) STBM interact with and activate monocytes. From the current data, we can rule out a role for IL-8 that is associated with eS20, or the proinflammatory cytokines IL-6, IL-1b and TNF-a, which are present only in trace concentrations in all STBM populations used in this study. However, it has been previously shown that STBM isolated by placental perfusion of the maternal circuit contained the biologically active isoform of IL-1b [31]. In contrast to the present work, where pS have been retrieved from placental washes lasting for 30 min, Di Santo and co-workers prepared STBM after 7 h of perfusion of the maternal compartment. On the other hand, we find that pS led to decreased CD11a expression on monocytes. CD11a forms together with CD18 the heterodimeric integrin lymphocyte function-associated antigen-1 (LFA-1). It has been suggested that monocytes strongly adhere to cultured syncytiotrophoblasts via LFA-1 [39]. We also have microscopic evidence that STBM bind to the cell surface of monocytes (Messerli, in preparation). Since STBM are likely to encompass the same membrane molecules as the parent cell from which they are derived, we would like to speculate that the observed changes in CD11a fluorescence intensity may be due to the interaction between the STBM and the monocytes through these partner molecules, resulting in some form of masking of the CD11a epitope to the antibody used for the present flow cytometric analysis. We are currently investigating this hypothesis. Other candidate
membrane molecules that signal through NF-kB and might mediate the STBM-induced proinflammatory response of monocytes are the toll-like receptors (TLRs) [40]. Recent work suggested that TLR responses in monocytes were impaired in pre-eclampsia [41]. We have previously shown that STBM are constituted of characteristic lipids and contain DNA molecules, which are all potential ligands of TLR [42,43]. In conclusion, our present analysis suggests that pS and eS20 bear dose-dependent proinflammatory properties for monocytes. This is interesting in regard to the in vivo situation in normal pregnancy, where the progressive monocytic activation in the maternal peripheral blood may be due to the expected increase in the load of placental microparticles with gestational age. Our results may also reflect the situation in pre-eclampsia, which is associated with enhanced circulatory STBM concentrations and overt monocyte activation. Furthermore, as the existing findings about a direct effect of STBM on endothelial cells remain inconclusive [24,44], we would like to propose that STBM-induced monocytes might, in a subsequent step, transmit the proinflammatory signal to the endothelium through their production of soluble and cell surface mediators of inflammation. Acknowledgements We are thankful to Dr Olav Lapaire, Dr Irene Hoesli and the clinical personnel of the Department of Obstetrics of the University Hospital, Basel, for their help in collecting placentas. We also thank Dr Dorothy Huang for her careful reading of the manuscript. Contract grant sponsor: the present work was supported in part by a grant from the Swiss National Science Foundation (3200B0107625). References [1] Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet 2005;365:785–99. [2] Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science 2005;308:1592–4. [3] Sacks G, Sargent I, Redman C. An innate view of human pregnancy. Immunol Today 1999;20:114–8. [4] Plum J, Thiery M, Sabbe L. Distribution of mononuclear cells during pregnancy. Clin Exp Immunol 1978;31:45–9. [5] Pitkin RM, Witte DL. Platelet and leukocyte counts in pregnancy. JAMA 1979;242:2696–8. [6] Koumandakis E, Koumandaki I, Kaklamani E, Sparos L, Aravantinos D, Trichopoulos D. Enhanced phagocytosis of mononuclear phagocytes in pregnancy. Br J Obstet Gynaecol 1986;93:1150–4. [7] Shibuya T, Izuchi K, Kuroiwa A, Okabe N, Shirakawa K. Study on nonspecific immunity in pregnant women: increased chemiluminescence response of peripheral blood phagocytes. Am J Reprod Immunol Microbiol 1987;15:19–23. [8] Luppi P, Haluszczak C, Betters D, Richard CA, Trucco M, DeLoia JA. Monocytes are progressively activated in the circulation of pregnant women. J Leukoc Biol 2002;72:874–84.
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